Sequestration of phosphoinositides by mutated MARCKS effector domain inhibits stimulated Ca(2+) mobilization and degranulation in mast cells

Abstract

Protein kinase C β (PKCβ) participates in antigen-stimulated mast cell degranulation mediated by the high-affinity receptor for immunoglobulin E, FcεRI, but the molecular basis is unclear. We investigated the hypothesis that the polybasic effector domain (ED) of the abundant intracellular substrate for protein kinase C known as myristoylated alanine-rich protein kinase C substrate (MARCKS) sequesters phosphoinositides at the inner leaflet of the plasma membrane until MARCKS dissociates after phosphorylation by activated PKC. Real-time fluorescence imaging confirms synchronization between stimulated oscillations of intracellular Ca(2+) concentrations and oscillatory association of PKCβ-enhanced green fluorescent protein with the plasma membrane. Similarly, MARCKS-ED tagged with monomeric red fluorescent protein undergoes antigen-stimulated oscillatory dissociation and rebinding to the plasma membrane with a time course that is synchronized with reversible plasma membrane association of PKCβ. We find that MARCKS-ED dissociation is prevented by mutation of four serine residues that are potential sites of phosphorylation by PKC. Cells expressing this mutated MARCKS-ED SA4 show delayed onset of antigen-stimulated Ca(2+) mobilization and substantial inhibition of granule exocytosis. Stimulation of degranulation by thapsigargin, which bypasses inositol 1,4,5-trisphosphate production, is also substantially reduced in the presence of MARCKS-ED SA4, but store-operated Ca(2+) entry is not inhibited. These results show the capacity of MARCKS-ED to regulate granule exocytosis in a PKC-dependent manner, consistent with regulated sequestration of phosphoinositides that mediate granule fusion at the plasma membrane.

FIGURE 1:

Dynamics of stimulated PKCβI-EGFP redistributions in IgE-sensitized RBL cells. (A) Time course of PKCβI-EGFP redistributions (green) in cells upon stimulation with antigen at 37°C (still frames from Supplemental Movie S1). Times in images specify intervals after antigen addition. Plot shows relative cytoplasmic fluorescence in the region of interest marked in a representative individual cell. Scale bar, 10 μm. (B) Time course of PKCβI-EGFP recruitment to the plasma membrane upon antigen stimulation at 25°C monitored as depletion of cytoplasmic fluorescence in a single representative cell. (C) Time course of PKCβI-EGFP recruitment to the plasma membrane upon stimulation with thapsigargin at 37°C monitored as depletion of cytoplasmic fluorescence for an average of six cells. (D) Time courses of PKCβI-EGFP recruitment to the plasma membrane monitored as depletion of cytoplasmic fluorescence (blue) and cytoplasmic [Ca²⁺] changes monitored by Fura Red (red) in a single representative cell stimulated with antigen at 37°C.

FIGURE 2:

Live-cell imaging of antigen-stimulated mRFP-MARCKS-ED and PKCβI-EGFP oscillations. (A) Representative images showing the distributions of PKCβI-EGFP (green) and mRFP-MARCKS-ED (red) before (top) and 100 s after antigen addition at 25°C (bottom) (still frames from Supplemental Movies S2a and S2b). Scale bar, 10 μm. (B) Time courses of cytoplasmic changes of PKCβI-EGFP (blue) and mRFP-MARCKS-ED (red) in a region of interest indicated in representative RBL cell in A.

FIGURE 3:

Association of mRFP-MARCKS-ED SA4 with the plasma membrane during stimulation by antigen. (A) Images of RBL cells transiently transfected with mutant mRFP-MARCKS-ED (red) and labeled with the [Ca²⁺] indicator Fluo-4 (green) at various times of stimulation by antigen at 25°C. Images show distributions of labels at time points before and after addition of antigen. Scale bar, 10 μm. (B) Fluorescence intensities from a cytoplasmic region of interest in a representative cell show that mRFP-MARCKS-ED SA4 (red) remains concentrated at the plasma membrane while stimulated [Ca²⁺] oscillations are observed. (C) Ca²⁺ mobilization in response to Ag at 25°C is delayed in a cell expressing mutant mRFP-MARCKS-ED (black) compared with an untransfected cell (blue); cells shown in Supplemental Movie S3. (D) Ca²⁺ response to antigen in the absence of extracellular Ca²⁺ is substantially delayed in a cell transiently expressing mutant mRFP-MARCKS-ED (red) compared with the response in an untransfected cell (blue) in the same sample.

FIGURE 4:

Antigen-stimulated Ca²⁺ mobilization is delayed in cells stably expressing mRFP-MARCKS-ED SA4. (A) Steady-state fluorimetry responses to antigen in control RBL cells (blue) and cells stably expressing mRFP-MARCKS-ED SA4 (red). (B) Ca²⁺ response to antigen in the absence of extracellular Ca²⁺ is substantially delayed in RBL cells stably expressing mutant mRFP-MARCKS-ED (red) compared with the response in untransfected cells (blue). Subsequent addition of 1.8 mM [Ca²⁺] results in similar Ca²⁺ entry for each. (C) Ca²⁺ mobilization in response to thapsigargin in RBL cells stably expressing mRFP-MARCKS-ED SA4 (red) is similar compared with the response in untransfected RBL cells (blue).

FIGURE 5:

MARCKS-ED SA4 inhibits antigen-stimulated degranulation monitored by real-time imaging. Degranulation monitored as FITC-dextran bursts from antigen-stimulated RBL cells shows delayed and reduced responses in cells expressing mRFP-MARCKS-ED SA4. Times in images indicate intervals after antigen addition; white arrow identifies a cell expressing mRFP-MARCKS-ED SA4. Scale bar, 10 μm. See Supplemental Movie S4.

FIGURE 6:

Inhibition of degranulation due to expression of MARCKS-ED SA4 as monitored by CD63 appearance at the plasma membrane. (A) Representative images showing reduced plasma membrane labeling by anti-CD63 due to antigen stimulation in cells expressing mutated MARCKS-ED expression. Scale bars, 10 μm. (B) Quantification of anti-CD63 labeling at the plasma membrane for unstimulated, antigen-stimulated, and thapsigargin-stimulated cells with and without expression of MARCKS-ED SA4. Error bars, SEM for 90–100 cells for each condition from n = 6 experiments. The p values from Student's t-test show **p < 0.01 for stimulated cells expressing MARCKS-ED SA4 compared with stimulated control cells.